Glycaemic control is associated with SARS-CoV-2 breakthrough infections in vaccinated patients with type 2 diabetes

Patients with type 2 diabetes (T2D) are characterized by blunted immune responses, which are affected by glycaemic control. Whether glycaemic control influences the response to COVID-19 vaccines and the incidence of SARS-CoV-2 breakthrough infections is unknown. Here we show that poor glycaemic control, assessed as mean HbA1c in the post-vaccination period, is associated with lower immune responses and an increased incidence of SARS-CoV-2 breakthrough infections in T2D patients vaccinated with mRNA-BNT162b2. We report data from a prospective observational study enroling healthcare and educator workers with T2D receiving the mRNA-BNT162b2 vaccine in Campania (Italy) and followed for one year (5 visits, follow-up 346 ± 49 days) after one full vaccination cycle. Considering the 494 subjects completing the study, patients with good glycaemic control (HbA1c one-year mean < 7%) show a higher virus-neutralizing antibody capacity and a better CD4 + T/cytokine response, compared with those with poor control (HbA1c one-year mean ≥ 7%). The one-year mean of HbA1c is linearly associated with the incidence of breakthrough infections (Beta = 0.068; 95% confidence interval [CI], 0.032-0.103; p < 0.001). The comparison of patients with poor and good glycaemic control through Cox regression also show an increased risk for patients with poor control (adjusted hazard ratio [HR], 0.261; 95% CI, 0.097-0.700; p = 0.008). Among other factors, only smoking (HR = 0.290, CI 0.146-0.576 for non-smokers; p < 0.001) and sex (HR = 0.105, CI 0.035-0.317 for females; p < 0.001) are significantly associated with the incidence of breakthrough infections.

The Paper by Marfella et al is of direct and immediate relevance it shows clearly for the first time that patients with diabetes and glyceamic control is associated with and significant increase in breakthrough infections in fully vaccination against Covid-19. This is based on some previous data of Israel but is nicely correlated in a prospective study with virus neutralising antibody titers and CD4+T/cytokine response. It confirms previous data showing that diabetes and especially purely controlled diabetes represents a form of an immunosuppressed condition. In their conclusions the authors should stress more clearly that diabetes care should be a key part of managing the current pandemic and post pandemic phase. This should also form a basis for the stratification of booster vaccinations and revaccinations in the high risk groups.
Reviewer #2 (Remarks to the Author): Marfella et al. report on the impact of glycaemic control on the response to COVID-19 vaccines and on the incidence of SARS-CoV-2 breakthrough infections. This study's results are supported by other former studies' findings. However, I find some major issues in this manuscript that should be addressed before publication: 1) The binary division to 2 classes, namely <7% and >7%, might be delusive and hide the effect of HbA1c levels on breakthrough infections rate. I would kindly suggest the authors re-analyzing the results with continuous variable of HbA1c levels. In addition, I would suggest plotting a figure of the distributions of HbA1c levels in the two groups. I could guess that looking at those distributions will show no justification to this partition (<7%, >7%).
2) As the authors show that the two groups are quite similar in most of their characteristics (except for their HbA1c), one could claim that these differences stem from the same reason: patients that are more careful are in better glycaemic control, and also more cautious about covid-19 and hence being more prone to be infected. Behavioral dissimilarities might dramatically affect the results, and without controlling them carefully I am afraid that these results are not reliable.
3) The study population size is limited and therefore conclusions regarding non-significant factors could mislead: finding variable to be non-significantly affecting is not the same as significantly finding it not affecting (or with negligible effect). minor: Line 90: "among those infected, COVID-19 can reach a mortality rate of 22%." -a meaningless sentence that seems to be out of context, and specifically not inferred from reference #3.
Combining the above 2nd and 3rd points, I think that concluding anything about vaccine effectiveness (breakthrough infections), which is vulnerable to behavior and other confounders, is difficult and almost impossible from a small population size.
In conclusion, this manuscript may be of interest to the scientific community. Nevertheless, before publication, the above points should be answered.

Response to reviewers' comments
We thank the referees and the editor for the helpful comments on the manuscript. We have addressed all the issues according to the reviewers' suggestions. We believe that these adjustments, elegantly suggested by the reviewers, have improved the paper in terms of clarity and accuracy. Changes are highlighted in the revised version of the manuscript. A clean copy is also attached.
We hope that the revised manuscript is suitable for publication in Nature Communications.

Point-by-point responses:
Reviewer #1 (Remarks to the Author):

This should also form a basis for the stratification of booster vaccinations and revaccinations in the high risk groups.
We really appreciate the positive evaluation provided by this Reviewer. In the revised version of the manuscript, we stressed the observation relative to diabetes care as a key part of the management of the pandemic and post-pandemic phase, advancing also the hypothesis that patients with poorly controlled diabetes might be included in the high-risk groups to be prioritised for booster vaccinations (page 10, lines 213,214, referring to the file with changes highlighted).

Marfella et al. report on the impact of glycaemic control on the response to COVID-19 vaccines and on the incidence of SARS-CoV-2 breakthrough infections. This study's results are supported by other former studies' findings. However, I find some major issues in this manuscript that should be addressed before publication:
We really thank the Reviewer for his/her thoughtful comments on our paper. These issues have been now addressed in the revised form of the manuscript.

The binary division to 2 classes, namely <7% and >7%, might be delusive and hide the effect of HbA1c levels on breakthrough infections rate. I would kindly suggest the authors re-analyzing the results with continuous variable of HbA1c levels. In addition, I would suggest plotting a figure of the distributions of HbA1c levels in the two groups. I could guess that looking at those distributions will show no justification to this partition (<7%, >7%).
We thank the Reviewer for the precious suggestion. We performed a linear regression to explore the association between the one-year mean of HbA1c, studied as a continuous variable, and breakthrough infections. The results showed a significantly increased risk with increasing HbA1c (Beta = 0.068; 95% confidence interval [CI], 0.032-0.103; p<0.001).
In other words, each increase in 1% of HbA1c was associated with an increased 6,8% (3,2% -10,3%) risk of experiencing a breakthrough infection. We modified the abstract, the methods and the results section to add this result (please see page 3, lines 47-49; and page 7, lines 136-139; and page 13, lines 306-308, referring to the file with changes highlighted).
In addition, to cope with the suggestion of this reviewer, we also showed the distribution of HbA1c values in the two groups of patients with vs those without infection (now Supplementary Figure 1). Results suggest that infected patients had significantly higher levels of the one-year mean of HbA1c (page 7, lines 145-147).

As the authors show that the two groups are quite similar in most of their characteristics (except for their HbA1c), one could claim that these differences stem from the same reason: patients that are more careful are in better glycaemic control, and also more cautious about covid-19 and hence being more prone to be infected. Behavioral dissimilarities might dramatically affect the results, and without controlling them carefully I am afraid that these results are not reliable.
We thank the Reviewer for raising a very important point. The study population was composed of healthcare and educator workers, whom theoretically should have the same caution in managing the risk of infection, given the stringent measures of prevention being applied in Italian hospitals and schools. Indeed, during the whole 2021, the use of face masks was mandatory everywhere except when outdoor (https://www.governo.it/it/articolo/domande-frequenti-sulle-misure-adottate-dalgoverno/15638#zone). In addition, the use of FFP2 masks was mandatory in hospitals and highly recommended for educators at school. On the other hand, as suggested by this Reviewer, it is hard to control for such behavioural dissimilarities among groups in observational studies. Thus, we added this aspect as a major limitation of the study (Discussion section: page 9, lines 187-189).
3. The study population size is limited and therefore conclusions regarding non-significant factors could mislead: finding variable to be non-significantly affecting is not the same as significantly finding it not affecting (or with negligible effect).
We agree with the Reviewer's observation. We added the limited sample size and the resulting impossibility to draw conclusions relative to non-significant variables as another limitation of the study (Discussion section: page 9, lines 189-192).

minor:
Line 90: "among those infected, COVID-19 can reach a mortality rate of 22%." -a meaningless sentence that seems to be out of context, and specifically not inferred from reference #3.
We are sorry for the oversight. We removed the suggested sentence.
We thank the reviewer for the precious suggestion. Accordingly, we re-performed the Cox regression analyses to include also sex as an additional covariate. The results show that also sex is significantly associated with breakthrough infections. These novel data and the modified results relative to the other variables have been added to the text, the figures, the table, and commented in the discussion section (please see Figure 4A, Table 1 We thank the reviewer for the helpful comment. Accordingly, we mentioned the observation that breakthrough infections are usually accompanied by a lower viral load (see page 9, lines 199-201), using the two suggested references. However, we have no data relative to the Ct of positive patients since nasopharyngeal swabs were processed in designated authoritative laboratories affiliated with the health national system but not in our lab. We mentioned this aspect as an additional limitation of the study (page 9, lines 199-201).
Combining the above 2nd and 3rd points, I think that concluding anything about vaccine effectiveness (breakthrough infections), which is vulnerable to behavior and other confounders, is difficult and almost impossible from a small population size.
In conclusion, this manuscript may be of interest to the scientific community.
Nevertheless, before publication, the above points should be answered.
We thank the reviewer for his/her overall evaluation and for the insightful comments. We believe that we addressed all the major issues suggested and that the paper is tangibly improved after the round of revision.